Differential regulation of high-affinity agonist binding to muscarinic sites in the rat heart, cerebellum, and cerebral cortex

The muscarinic agonist [3H]cismethyldioxolane ([3H]CD) was used to characterize the effects of regulators upon high-affinity agonist binding sites of the rat heart, cerebral cortex and cerebellum. Comparative studies with sodium ions (Na+), magnesium ions (Mg++), N-ethylmaleimide (NEM) and the guanine nucleotide Gpp(NH)p revealed tissue-specific effects. Mg++ preferentially enhanced while Gpp(NH)p and NEM reduced high-affinity [3H]CD binding in the heart and cerebellum. By comparison NEM enhanced high-affinity agonist binding in the cerebral cortex while Gpp(NH)p and Mg++ had little or no effect. Kinetic studies support an allosteric mechanism for these effects and provide further evidence for muscarinic receptor subtypes in mammalian tissues.     

Decreased ADP-ribosylation of the Galpha(olf) and Galpha(s) subunits by high glucose in pancreatic B-cells

In HIT-T15 insulinoma B-cells incubated in presence of [(32)P]NAD, we identified by autoradiography and immunoblotting ADP-ribosylation (ADP-R) of the trimeric G-protein Galpha(s) and Galpha(olf) subunits (45 kDa) induced by cholera toxin in M1 (120,000g) and M2 (70,000g) subcellular fractions containing plasma membranes, insulin granules, and mitochondria. This ADP-R indicates that these two fractions contain functionally competent Galpha subunits for adenylyl cyclase activation. Prolonged exposure of HIT-T15 cells to high glucose (25 mM instead of 6 mM) specifically reduced the ADP-R in Galpha(s) and Galpha(olf) subunits in the M1 fraction only, despite the clear increase of their accumulation in this compartment. A similar alteration in the ADP-R of the M1-associated Galpha(s) and Galpha(olf) subunits was observed in pancreatic islets isolated from fasted and fed rats. These results may explain, at least in part, the undesirable effects of sustained hyperglycemia on the cAMP-dependent process of insulin secretion in diabetes.     

Multiple interconvertible affinity states for the delta opioid agonist-receptor complex

Previously we demonstrated that rates of dissociation of [3H-D-Ala2-D-Leu5]enkephalin [( 3H]DADL) from bovine hippocampal synaptic plasma membranes (SPMs) varied depending upon association time, suggesting a multistep binding process. To characterize different kinetic intermediates, we examined the effects of guanine nucleotide on dissociation rate. Control off-rates were compared to those obtained when guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) (50 microM) was added either coincident with the radioligand at association or with 1 microM unlabeled DADL which initiated dissociation. delta site selectivity of [3H]DADL was ensured by addition of 20 nM unlabeled [D-Ala2-Me-Phe4-Gly-Ol5]enkephalin which suppressed mu site cross-reactivity in this preparation. Addition of Gpp(NH)p at the onset of dissociation increased the off-rate to a much greater extent after steady state binding was reached (60 min) compared to that following an association time of only 7 or 20 min. A slowly formed high affinity state appeared to be rapidly converted to a lower affinity state under these conditions. When Gpp(NH)p was present throughout the association period, the slowly dissociating state was no longer observed. Also, the off-rate following a 7-min association is linear and much faster than control, suggesting that Gpp(NH)p may affect an initial intermediate state as well as the high affinity complex. Pretreatment of the membranes with N-ethylmaleimide (NEM) eliminated the association time-dependent dissociation rates, apparently preventing time-dependent formation of a high affinity state. This state is thought to be possibly a ligand-receptor complex interacting with a GTP binding protein. However, the rate of dissociation from NEM-treated membranes was accelerated by addition of Gpp(NH)p and the effect was not association time-dependent. NEM treatment resulted in an increased potency for Gpp(NH)p inhibition of [3H]DADL steady state binding. These results suggest the occurrence of at least three steps in the association of DADL to bovine hippocampal synaptic membranes.     

Vicinal dithiol-disulfide distribution in the Escherichia coli mannitol specific carrier enzyme IImtl

Escherichia coli mannitol specific EII in membrane vesicles can be inhibited by the action of the oxidizable substrate-reduced phenazine methosulfate (PMS) in a manner similar to E. coli enzyme IIGlc [Robillard, G. T., & Konings, W. (1981) Biochemistry 20, 5025-5032]. The fact that reduced PMS and various oxidizing agents protect the enzyme from inactivation by the sulfhydryl reagents N-ethylmaleimide and bromopyruvate suggests that the active form possesses a dithiol which can be protected by conversion to a disulfide. The sulfhydryl-disulfide distribution has been examined in purified EIImtl by labeling studies with N-[1-14C]ethylmaleimide ( [14C]NEM). EIImtl can be alkylated at three positions per peptide chain. When alkylation takes place in 8 M urea, only two positions are labeled. The third position becomes labeled in urea only after treatment with DTT, suggesting that the native enzyme is composed of two subunits linked by a disulfide bridge. The remaining two sulfhydryl groups per peptide chain appear to undergo changes in oxidation state as indicated by the following results. (1) Treatment of the active enzyme with NEM leads to complete inactivation and incorporation of 1 mol of [14C]NEM per peptide chain. Oxidizing agents protect the activity and prevent labeling presumably by forming a disulfide. (2) Phosphorylating the enzyme (one phosphoryl group per peptide chain) fully protects the activity, but 1 mol of NEM per peptide chain is still incorporated. Subsequent dephosphorylation by adding mannitol causes a second mole of [14C]NEM to be incorporated and results in complete inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding

We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding. A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond. This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH. This is based on the comparative tryptic peptide mapping of [14C]NEM-modified p21 in the presence and absence of GTP. The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence. Amino acid analysis of the purified [14C]NEM-modified peptide from tryptic digests of p21 also confirmed its identity. This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins.     

Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.     

Characterization of transducin from bovine retinal rod outer segments. The role of sulfhydryl groups

The properties and functions of the sulfhydryl groups of transducin were examined by 5,5' -dithiobis-(2-nitrobenzoic acid) titration and N-ethylmaleimide modification. The T beta gamma subunit of transducin contained a total of six free sulfhydryl groups and two were reactive under native conditions. Both reactive sulfhydryl groups were located in the beta polypeptide. The functions of transducin were not affected by the modification of these two sulfhydryl groups. The T alpha subunit of transducin contained three accessible sulfhydryl groups under both native and denaturing conditions. When 1.3 sulfhydryl groups were covalently modified by N-ethylmaleimide, the GTPase activity, the guanosine 5' -(beta, gamma-imido)triphosphate (Gpp(NH)p) uptake, and the rhodopsin-binding property of transducin were inhibited. The binding of Gpp(NH)p to T alpha blocked two of the three sulfhydryl groups from chemical modification and increased the reactivity of the remaining one. Modification of this specific sulfhydryl group of T alpha -Gpp(NH)p inhibited the exchange of the bound Gpp(NH)p for GTP. However, the modified T alpha-Gpp(NH)p was able to activate cGMP phosphodiesterase in solution and on positively charged liposomes. These findings demonstrated that a conformational change of T alpha occurs upon the binding of Gpp(NH)p and a specific sulfhydryl group of T alpha plays an important role in the activation of transducin in retinal rod outer segments.     

Chemical modification of pig liver initiation factor eIF-2 with N-ethylmaleimide. Amino acid sequences around the N-ethylmaleimide-reactive sulfhydryl groups and the effect of GDP on the modification

The activity of eukaryotic initiation factor eIF-2 as to the formation of the ternary complex, eIF-2 GTP Met-tRNA(f), is inhibited by N-ethylmaleimide. Our preparation of pig liver eIF-2 contained alpha and gamma subunits and was inhibited by more than 90% by N-ethylmaleimide. Using our eIF-2, we determined the sequences around the N-ethylmaleimide-reactive sulfhydryl groups, studied the effect of GDP on the sulfhydryl modification and that of NEM on the [3H]GDP binding, and examined the protective effect of GTP against the inhibition of ternary complex formation by N-ethylmaleimide. Both subunits of native eIF-2 contained [14C]N-ethylmaleimide-reactive sulfhydryl groups. One N-ethylmaleimide-reactive sulfhydryl group was in the alpha subunit and 4 were in the gamma subunit. The sequence of the peptide of the alpha subunit was determined to be: Ala-Gly-Leu-Asn-Cys-Ser-Thr-Glu-Thr-Met-Pro-Ile. Two of the four [14C]N-ethylmaleimide-reactive sulfhydryl groups in the gamma subunit were highly reactive, their sequences being: Ile-Val-Leu-Thr-Asn-Pro-Val-Cys-Thr-Glu-Val-Gly-Glu-Lys (gamma 1); Ser-Cys-Gly-Ser-Ser-Thr-Pro-Asp-Glu-Phe-Pro-Thr-Asp-Ile-Pro-Gly-Thr-Lys (gamma 3a). Peptide gamma 3a contained the consensus sequence element (AspXaaXaaGly) of GTP-binding proteins. With preincubation of eIF-2 with GDP, the incorporation of [14C]N-ethylmaleimide into the gamma subunit was reduced to 40% of the control level, but the 14C-incorporation into the alpha subunit did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of polyoma virus middle T antigen-associated tyrosyl kinase activity by N-ethylmaleimide

The middle T antigen (MT Ag) encoded by polyoma virus has an associated protein kinase activity which transfers a phosphoryl group from ATP or GTP to a tyrosine residue on MT Ag in immunoprecipitates formed between polyoma virus-infected or transformed cell extracts and serum from animals bearing polyoma-induced tumors. Incubation of such immunoprecipitates or polyoma-transformed cell extracts prior to immunoprecipitation with the sulfhydryl reagent, N-ethylmaleimide (NEM), resulted in a significant inhibition of MT Ag-associated kinase activity. Inactivation of this enzyme activity by NEM was found to be dependent upon the incubation pH, time of incubation, and NEM concentration. ATP, GTP, and ADP in the presence of Mg2+ were found to decrease the rate of NEM-mediated inactivation of MT Ag-associated kinase activity, while CTP and UTP did not detectably alter the rate of enzyme inhibition by NEM. These results suggest that the MT Ag-associated kinase possesses at least one NEM-sensitive sulfhydryl group essential for phosphotransferase activity which may be present at or near the enzyme catalytic site.     

Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide

Pig heart NADP-specific isocitrate dehydrogenase is inactivated by N-ethylmaleimide (NEM) (Colman, R. F., and Chu, R. (1970) J. Biol. Chem. 245, 601-607), and is completely protected against inactivation, but not against the incorporation of NEM, by isocitrate plus Mn2+. We have now treated the enzyme with [3H]NEM in the absence and presence of isocitrate plus Mn2+, digested it with trypsin, and isolated and sequenced the labeled Cys peptides. In the inactive enzyme, two major peptides, SSGGFVWACK and DLAGCIHGLSNVK, and two minor peptides, CATITPDEAR and EPIICK, were labeled at Cys. Upon reaction with [3H]NEM in the presence of isocitrate plus Mn2+, full catalytic activity was retained and only DLAGCIHGLSNVK was labeled; the Cys of this peptide is therefore not essential for catalysis. The modification of SSGGFVWACK appears to be the major cause of inactivation by NEM. The Cys in SSGGFVWACK may have a catalytic role, most likely in the strengthened binding of Mn2+ in the presence of isocitrate. Isocitrate dehydrogenase was carboxymethylated under denaturing conditions with [14C]iodoacetate and digested with trypsin; 6 unique labeled Cys peptides, containing 6 unique Cys residues, were purified and sequenced. Six corresponding peptides were isolated from enzyme treated under denaturing conditions with [3H]NEM. These results eliminate the previous uncertainty regarding the number of Cys residues in the enzyme. A comparison of the sequences of the NH2-terminal 30 residues and the 6 Cys peptides of the pig heart NADP-dependent isocitrate dehydrogenase with the Escherichia coli NADP enzyme provides evidence for great dissimilarity between the two enzymes.     

Mechanism of adenylate cyclase activation by the rat lung cytoplasmic factors

Epinephrine, histamine and prostaglandin E₁ stimulated adenylate cyclase activity in lung membranes and their stimulation of the enzyme activity was completely blocked by propranolol, metiamide and indomethacin, respectively. A partially-purified activator from the adult rat lung also enhanced adenylate cyclase activity in membranes. However, stimulation of adenylate cyclase by the rat lung activator was not abolished by the above receptor antagonists. Further, epinephrine, NaF and Gpp(NH)p stimulated adenylate cyclase activity rather readily, whereas stimulation of the enzyme activity by the lung activator was evident after an initial lag phase of 10 min. Also, the lung activator produced additive activation of adenylate cyclase with epinephrine, NaF and Gpp(NH)p. These results indicate that the lung activator potentiates adenylate cyclase activity in membranes by a mechanism independent from those known for epinephrine, NaF and Gpp(NH)p. Incubation of lung membranes for 30 min at 40°C resulted in a loss of adenylate cyclase activation by NaF and Gpp(NH)p. Addition of the released proteins to the heat-treated membranes did not restore the enzyme response to these agonists. However, heat treatment of lung membranes in the presence of 2-mercaptoethanol or dithiothreitol prevented the loss of adenylate cyclase response to NaF and Gpp (NH)p. N-ethylmaleimide abolished adenylate cyclase activation by epinephrine, NaF, Gpp(NH)p and the lung activator. These results indicate that the sulfhydryl groups are important for adenylate cyclase function in rat lung membranes.Abbreviations Gpp(NH)p 5-Guanylimidodiphosphate     

Characteristics of an adenosine A1 binding site in human placental membranes

Binding sites were solubilized from human placental membrane using 1.5% sodium cholate and were assayed using polyethylene glycol precipitation. These soluble binding sites had properties of an adenosine A1 binding site. 2-[3H]Chloroadenosine and N-[3H]-ethylcarboxamidoadenosine (NECA) binding were time dependent and reversible. Scatchard plots indicate two classes of binding sites with Kd values of 6 and 357 nM for 2-chloro[8-3H]adenosine and 0.1 and 26 nM with [3H]NECA. The specificity of [3H]NECA binding was assessed by the ability of adenosine analogs to complete for binding sites. Using this approach the estimated IC50 values were 60 nM for (R-PIA), 160 nM for S-PIA, 80 nM for NECA, and 20 nM for 2-chloroadenosine. Binding of [3H]NECA to the soluble sites is inhibited to 48% of the control value by 100 microM guanylyl-5'-imidodiphosphate (Gpp(NH)p). The IC50 value for NECA binding to the soluble binding site was increased from 80 nM to 1500 by Gpp(NH)p. There was a shift of binding affinity from a mixture of high and low affinity to only low affinity with 100 microM Gpp(NH)p. Despite these alterations a NECA prelabeled molecular species of 150 kDa did not decrease in molecular weight upon the addition of 100 microM Gpp(NH)p during high-performance liquid chromatography on a Superose 12 column. Other evidence to support the concept of preferential solubilization and assay of a small population of A1 binding sites was obtained. Following solubilization adenosine A2-like binding sites could be detected only in reconstituted vesicles. The existence of small amounts of A1 binding sites in intact human placental membranes was directly demonstrated using the A1 agonist ligand N6-[3H]cyclohexyladenosine and the A1 antagonist ligand 8-[3H]cyclopentyl-1,3-dipropylxanthine. JAR choriocarcinoma cells have "A2-like" membrane binding sites. In contrast to placental membranes, only A2-like binding sites could be solubilized from JAR choriocarcinoma cells. These observations indicate that human placental membranes contain adenosine A1 binding sites in addition to A2-like binding sites. These sites are guanine nucleotide sensitive, but do not shift to a lower molecular weight form upon assumption of a low affinity state.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

Affinity labeling of delta-opiate receptors using [D-Ala2,Leu5,Cys6]enkephalin. Covalent attachment via thiol-disulfide exchange

[D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is a synthetic enkephalin analog which contains a sulfhydryl group. DALCE binds with high affinity to delta-receptors, with moderate affinity to mu-receptors, and with negligible affinity to kappa-receptors. Pretreatment of rat brain membranes with DALCE resulted in concentration-dependent loss of delta-binding sites. Using 2 nM [3H][D-Pen2,D-Pen5]enkephalin (where Pen represents penicillamine) to label delta-sites, 50% loss of sites occurred at about 3 microM DALCE. Loss of sites was not reversed by subsequent incubation in buffer containing 250 mM NaCl and 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), conditions which cause dissociation of opiate agonists. By contrast, the enkephalin analogs [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, [D-Pen2,D-Pen5]enkephalin, and [D-Ala2,D-Leu5,Lys6]enkephalin were readily dissociated by NaCl and Gpp(NH)p, producing negligible loss at 3 microM. This suggests that DALCE binds covalently to the receptors. Pretreatment of membranes with the reducing agents dithiothreitol and beta-mercaptoethanol had no effect on opiate binding. Thus, loss of sites required both specific recognition by opiate receptors and a thiol group. The irreversible effect of DALCE was completely selective for delta-receptors. Pretreatment with DALCE had no effect on binding of ligands to mu- or kappa-receptors. The effect of DALCE on delta-binding was: 1) markedly attenuated by inclusion of dithiothreitol in the preincubation buffer, 2) partially reversed by subsequent incubation with dithiothreitol, 3) slightly enhanced when converted to the disulfide-linked dimer, and 4) prevented by blocking the DALCE sulfhydryl group with N-ethylmaleimide or iodoacetamide. These results indicate that DALCE binds covalently to delta-receptors by forming a disulfide bond with a sulfhydryl group in the binding site. The mechanism may involve a thiol-disulfide exchange reaction.     

MgCl2-sensitive and Gpp(NH)p-sensitive antagonist binding states of rat heart muscarinic receptors: preferential detection at ambient temperature assay and location in two subcellular fractions

Summary Some novel observations dealing with antagonist binding to cardiac particulate muscarinic receptors are described. Gpp(NH)p increased (2–3 fold) the specific binding of [³H]-QNB or [³H]-NMS, both potent muscarinic antagonists, to washed particles (WP), but not microsomes (MIC), when the binding was conducted at 30°C. Magnesium, on the other hand, increased (2–3 fold) the binding of these antagonists to MIC, but not to WP, under the same condition. The treatment of subcellular fractions with 0.2 mM N-ethylmaleimide (NEM), a sulfhydryl reagent, failed to significantly modify the respective stimulatory actions of either Gpp(NH)p on WP binding or of magnesium on MIC binding of these antagonists; treatment with dithiothreitol (1 mM) was also ineffective in this regard. Gpp(NH)p decreased K_d (WP) while magnesium increased K_d (MIC) for [³H]-QNB. Repeated freezing/thawing of isolated subcellular fractions abolished the stimulatory effect of magnesium on onist binding to MIC but not of Gpp(NH)p on WP antagonist binding; the freeze/thaw procedure per se increased MIC binding but not WP binding of these antagonists. When the binding was conducted at 4°C (24 hr), the stimulatory effect of Gpp(NH)p on [³H]-QNB binding was enhanced (6-fold) in the case of WP and was detectable (80%) in the case of MIC. Under this condition, the stimulatory effect of magnesium on [³H]-QNB binding was also enhanced (5-fold) in the case of MIC and became evident (200%) in the case of WP. The results of this work support the following views: (a) antagonist-occupied cardiac muscarinic receptors are capable of interaction with guanine nucleotide binding proteins (G protein like G₁,G_o) and such interaction influences antagonist binding properties (e.g. increased affinity) of the cardiac membrane-associated muscarinic receptors (b) magnesium influences (decreased affinity) antagonist binding properties by interacting with multiple sites of which some are likely associated with components other than G proteins of the particulate fractions (c) a pool of NEM-sensitive sulfhydryls involved in the regulation of Gpp(NH)p-sensitive agonist binding to cardiac muscarinic receptors is not involved in the regulation by either Gpp(NH)p or magnesium of antagonist binding in these subcellular fractions and (d) membrane fluidity and microenvironment surrounding the receptor and G proteins contribute to the actions of Gpp(NH)p and magnesium on antagonist binding.     

Selective Protection of Benzomorphan Binding Sites Against Inactivation by N-Ethylmaleimide. Evidence for k-Opioid Receptors in Frog Brain

Selective binding of [3H]bremazocine and [3H]-ethylketocyclazocine to kappa-opioid receptor sites in frog (Rana esculenta) brain membranes is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide (NEM). Pretreatment of the membranes with kappa-selective compounds [ethylketocyclazocine (EKC), dynorphin (1-13), or U-50,488H] but not with [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO; mu specific ligand) or [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DADLE; delta specific ligand) strongly protects the binding of the radioligands against NEM inactivation. These results provide more evidence for the existence of kappa-opioid receptors in frog brain. The relatively high concentrations of NEM that are needed to decrease the specific binding of [3H]bremazocine together with the observation of an almost complete protection of its binding sites by NaCl suggest that bremazocine may act as an opioid antagonist in frog brain.     

Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H] CD). Characterization studies on rat forebrain homogenates showed that [³H] CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [³H] CD binding in the rat brain correlated generally with [³H] (?)3-quinuclidinyl benzilate ([³H] (?)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], caused a 43–77% inhibition of [³H] CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H] CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H] CD binding, Gpp(NH)p caused an increase in [³H] (?)QNB binding in the heart heart and ileum and no change in [³H] (?)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H] (?)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [³H] CD binding by NEM.     

Evidence for specific binding sites for guanine nucleotides in adipocyte and hepatocyte plasma membranes. A difference in fate of GTP and guanosine 5'-(beta, gamma-imino) triphosphate.

Binding and degradation of GTP and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p by plasma membranes from rat liver and fat cells were investigated. Gpp(NH)p is hydrolyzed predominantly by nucleotide pyrophosphohydrolases in the membranes, whereas GTP is hydrolyzed primarily by nucleotide phosphohydrolases. These enzymes are not specific for the guanine nucleotides since co-addition of the analogous adenine nucleotides spares their hydrolysis. Both Gpp(NH)p and GTP are taken up by the membranes at sites which, to the extent that high concentrations of the corresponding adenine nucleotides fail to inhibit uptake, appear to be specific for guanine nucleotides. Gpp(NH)p taken up at these sites remains essentially intact irrespective of the degree of hydrolysis of unbound Gpp(NH)p by nucleotide pyrophosphohydrolases, indicating that the binding siteis incapable of degrading Gpp(NH)p. GTP and GDP inhibit competitively the binding of Gpp(NH)p; the binding constants for the three nucleotides are similar (0.1 to 0.4 muM) and are in the same range required for their effects on adenylate cyclase activity. Binding of the nucleotides is inhibited by sulfhydryl agents, suggesting that a sulfhydryl group is involved in the binding process. In contrast to binding of Gpp(NH)p, uptake of GTP is accompanied by substantial hydrolysis, primarily to GDP, under incubation conditions (high [ATP] plus ATP regenerating system) in which [GTP] in the medium remains essentially constant. GDP bound to the membranes is progressively hydrolyzed to 5'-GMP. Thus, GTP and Gpp(NH)p, although binding to the same specific sites, are differentially susceptible to hydrolysis at their terminal phosphates when bound to these sites. These findings are discussed in terms of the markedly different potencies of GTP and Gpp(NH)p as activators of adenylate cyclase systems.     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Mechanism of action of ATP on intestinal epithelial cells. Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH2)p. ATP also increased the formation of cyclic [14C]AMP in enterocytes that had been preincubated with [14C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and augmenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the Isc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.